The present invention relates to a method and apparatus for producing microarrays. More specifically, the present invention relates to a method and apparatus for increasing the speed of production of compact microarrays.
As is well known (and described for example in U.S. Pat. No. 5,807,522 to Brown et al. and in xe2x80x9cDNA Microarrays: A Practical Approachxe2x80x9d, Schena, Mark, New York, Oxford University Press,1999, ISBN 0-19-963776-8), microarrays are arrays of very small samples of purified DNA or protein target material arranged as a grid of hundreds or thousands of small spots on a solid substrate. When the microarray is exposed to selected probe material, the probe material selectively binds to the target spots only where complementary bonding sites occur, through a process called hybridization. Subsequent quantitative scanning in a fluorescent microarray scanner may be used to produce a pixel map of fluorescent intensities (See, e.g., U.S. Pat. No. 5,895,915, to DeWeerd et al.). This fluorescent intensity map can then be analyzed by special purpose quantitation algorithms which reveal the relative concentrations of the fluorescent probes and hence the level of gene expression, protein concentration, etc., present in the cells from which the probe samples were extracted.
The microarray substrate is generally made of glass which has been treated chemically to provide for molecular attachment of the spot samples of microarray target material. The microarray substrate is also generally of the same size and shape as a standard microscope slide, about 25 mmxc3x9775 mmxc3x971 mm thick. The array area can extend to within about 1.5 mm of the edges of the substrate, or can be smaller. The spots of target material (typically DNA) are approximately round. The spot diameter is generally determined by the dispensing or spotting technique used and typically varies from about 75 microns to about 500 microns, and may be as small as about 20 microns. The general trend is toward smaller spots, which produce more compact arrays. The center-to-center spacing between the spots usually falls into the range of 1.5 to 2.5 spot diameters.
FIG. 1A, which is not drawn to scale, shows a top view of a prior art microarray 100. In FIG. 1A, each of the circles represents a tiny spot of target material that has been deposited onto a rectangular glass substrate 101, and the spots are shown in a magnified view as compared to the substrate 101. Assuming typical dimensions of 100 xcexcm spot diameter and 200 xcexcm centerto-center spacing between the spots, the illustrated six by six array of spots covers only a 1100 xcexcm by 1100 xcexcm square area of the 25 mm by 75 mm area defined by the substrate 101. Thousands of spots are usually deposited in a typical microarray and the spots may cover nearly the entire substrate. The portion of the microarray that is covered with spots of target material may be referred to as the xe2x80x9cactive areaxe2x80x9d of the microarray.
There are several well known methods of depositing the spots onto the substrate of a microarray, and instruments that deposit the spots are typically referred to as xe2x80x9cspotting instrumentsxe2x80x9d. One popular method is to use one or more xe2x80x9cpinsxe2x80x9d to transfer the target material from a reservoir onto the microarray substrate. FIG. 1B shows an example of such a prior art pin 102, which includes a pin head 104 and a needle 106. Both the pin head 104 and the needle 106 are generally cylindrical, and the pin head 104 and needle 106 are generally disposed so that they are coaxial. The diameter of the pin head 104 is greater than the diameter of the needle 106, and the needle is substantially longer than the pin. One end 107 of the needle 106 is tapered or sharpened, and the other end of the needle is attached or bonded to the pin head 104. Examples of such pins are described in, for example, U.S. Pat. Nos. 5,770,151 (Roach et al.) and U.S. Pat. No. 5,807,522 (Brown et al.).
In operation, the sharp ends 107 of the pins are dipped into a reservoir of the liquid target material so that some of the material is xe2x80x9cpicked upxe2x80x9d or becomes attached to the pins. The sharp ends of the pins are then placed in contact with the substrate to deposit tiny amounts of the material onto selected locations of the substrate. The pins are normally moved by a mechanical or robotic apparatus so the spots may be accurately placed at desired locations on the substrate.
Some types of pins are capable of absorbing only enough target material to form a single spot on the microarray before they need to be re-dipped in the reservoir, whereas others can absorb eriough target material from the reservoir to form several or even hundreds of spots before they need to be re-dipped in the reservoir. In either case, the pins must be manufactured to very precise tolerances to insure that each spot formed by the pin will be of controlled size. As a result of these demanding specifications, the pins are rather expensive (e.g., a single pin typically costs several hundred dollars). Also, the sharp ends of the pins are so small and precisely shaped (e.g., a square tip measuring 50 microns on a side) that the pins are fragile. Accordingly, to prevent damage, the sharp ends of the pins must only be exposed to a tiny force when the sharp ends are placed in contact with the substrate or any other solid object.
Spotting instruments typically form microarrays in batches. For example, in a single xe2x80x9crunxe2x80x9d, a spotting instrument may form up to 100 identical microarrays. After forming enough spots to complete the batch of microarrays being spotted, the pins generally need to be washed (to remove any excess liquid target material), and then dried before they can be dipped into another reservoir of target material. So the process of forming microarrays with a xe2x80x9cpin-typexe2x80x9d spotting instrument includes steps of (1) positioning a pin over a reservoir of target material; (2) dipping the sharp end of the pin into the reservoir; (3) withdrawing the sharp end of the pin from the reservoir; (4) moving the pin over a selected location within the active area of a microarray; (5) lowering the pin to bring the sharp end of the pin into contact with the microarray substrate to form a single spot of controlled size at the selected location; (6) raising the pin to separate the sharp end of the pin from the substrate; (7) repeating steps (4), (5), and (6) until the pin""s supply of target material is exhausted or until the desired number of spots have been placed on the bach of microarrays being produced; (8) washing the pin by either placing the pin in a stream of cleaning solution or by dipping the pin into a reservoir of cleaning solution; and (9) drying the pin. The spotting instrument repeats all of these steps numerous times to form a single microarray.
Since microarrays typically include thousands of spots, using only a single pin to form the microarray would be extremely time consuming. Accordingly, spotting instruments are often capable of simultaneously manipulating several pins. FIGS. 1C, 1D and 1E show side, top, and perspective, views respectively of a printhead 110 that can simultaneously hold sixteen pins 102. Printhead 110 is a solid block of material, typically metal, that defines an array of sixteen apertures 112. The apertures 112 are slightly larger than the outer diameter of the needles 106 so the needles can extend through the apertures 112. The apertures 112 are also smaller than the outer diameter of the pin heads 104 so that when the needle of a pin is dropped into one of the apertures 112, the pin head 104 will be supported by the upper surface of the printhead 110. The pins are thereby xe2x80x9cslip-fitxe2x80x9d into the apertures of the printhead. FIGS. 1F and 1G show side and top views, respectively, of sixteen pins mounted into printhead 110.
FIG. 1H illustrates printhead 110 being lowered to place the sharp ends of the pins 102 into contact with substrate 101 and thereby simultaneously forming sixteen spots of target material on the substrate. As shown, the printhead is generally lowered about 1 mm further than required to place the sharp ends of the pins in contact with the substrate. The slip-fit allows the upper surface of the printhead to be lowered beneath the bottom of the pin heads without imparting significant force to the sharp ends of the pins. The printhead is preferably lowered sufficiently slowly so that the force applied to the sharp ends of the pins (1) is principally determined by the weight of the pin plus a minor additional force due to the friction of the slip-fit and (2) is not significantly affected by inertial forces. The act of lowering the printhead to place the sharp ends of the pins in contact with the substrate and thereby forming spots on the microarray is commonly referred to as xe2x80x9cprintingxe2x80x9d.
Commercially available printheads provide between 4 and 72 apertures, thereby accommodating between 4 and 72 pins. Commercially available reservoirs provide a plurality of wells, or individual reservoirs, and permit each pin mounted in a printhead to be dipped into a separate well. Two popular reservoirs useful for producing microarrays are the xe2x80x9c96-well platexe2x80x9d and the xe2x80x9c384-well platexe2x80x9d. Each of these plates provide a rectangular array of wells, each well being capable of holding a unique sample of liquid target material. FIG. 1I shows a top view of a 96-well plate. In 96-well plates, the centers of the individual reservoirs are separated by 9.0 mm, and in 384-well plates, the centers of the individual reservoirs are separated by 4.5 mm. The centers of adjacent apertures in commercially available printheads are correspondingly separated by either 9.0 or 4.5 mm. Pin-type spotting instruments generally include mechanisms for holding or manipulating one or more plates (e.g., either 96-well or 384-well), a printhead, a robotic manipulator for controlling the movement of the printhead, mechanisms for holding a plurality of substrates, a pin washer, and a dryer.
FIGS. 2A and 2B illustrate some of the steps in forming a microarray 200 with a pin-type spotting instrument. Microarray 200 has a rectangular substrate 101 measuring 75 mm by 25 mm and a rectangular active area 202 (within which all spots will be deposited) measuring 18 mm by 54 mm. FIG. 2A shows a rectangular area 210 within which 48 spots have been deposited using a printhead carrying 48 pins. The area within which the 48 spots have been placed is called the xe2x80x9cfootprintxe2x80x9d 210 of the printhead. A 48-pin printhead, carrying four rows of pins with twelve pins in each row, in which the centers of the pins are spaced apart by 4.5 mm, has a rectangular xe2x80x9cfootprintxe2x80x9d that measures 13.5 mm by 49.5 mm, where the xe2x80x9cfootprintxe2x80x9d is the minimum (regularly-shaped, contiguous) area that contains all the spots made by allowing all the pins in the printhead to contact the substrate once. In other words, every single printing deposits 48 spots of target material onto the substrate and those 48 spots fit into a rectangular 13.5 mm by 49.5 mm footprint.
FIG. 2A shows a top view of the footprint 210 of such a printhead superposed onto substrate 101. Footprint 210 represents an area within which 48 spots have been printed onto the substrate 101 of a microarray. Since each spot has a diameter of only about 20 to 500 microns, and since footprint 210 includes only 48 spots, most of the footprint 210 is occupied by empty space (i.e., most of the area of the footprint 210 is not occupied by spots of target material). The microarray is created by repeatedly printing spots onto the substrate with the footprint of each printing being slightly offset (and mostly overlapping) with the footprints associated with all the other printings. FIG. 2B shows the footprint 212 of a second printing. The combined areas of the two footprints 210, 212 will contain 96 spots: two arrays of 48 spots with each array being slightly offset from the other. The microarray is created by repeatedly printing arrays of spots until all desired spots have been placed on the substrate. Normally, the active area of the microarray is filled in until any additional printings would form spots that overlie or otherwise disturb spots from previous printings. The active area generated by the above-discussed 48-pin printhead is typically 18 mm by 54 mm.
One obvious advantage of using multiple pins, is that it reduces the number of printing steps, and therefore the time, required to produce a microarray. However, one disadvantage of using multiple pins is that it increases the difficulty of making a xe2x80x9ccompactxe2x80x9d microarray. For example, as shown in FIGS. 2A and 2B, the active area of the microarray (i.e., the area that contains all the spots) must be at least as large as the printhead""s footprint. Since the size of the footprint increases with the addition of more pins, increasing the number of pins used tends to increase the overall size of the active area of the microarray.
Decreasing the active area of a microarray, or making the microarray more xe2x80x9ccompactxe2x80x9d, has several advantages. First, the hybridization reaction requires less fluorescently labeled probe material if the active area of the microarray is small, and the probe material is expensive and technically difficult to make in large volumes. Second, the hybridization reaction is more likely to be uniform in rate and extent over a smaller array area than over a large area, producing results of higher quality. Third, subsequent to hybridization, the microarrays are normally scanned for quantitative fluorescence intensity. The output of this scanning process is image files, at least two files per microarray, each file typically of several megabytes. These image files are used as inputs to microarray quantification software programs, which extract numerical results from them. More compact arrays produce smaller image files, easing the downstream data storage and image quantification processes. The relative importance of each of these factors varies from application to application, and experiment to experiment.
By way of example, one useful xe2x80x9ccompactxe2x80x9d microarray configuration has an active area that is an 18 mm square, and 20,000 spots of 80 xcexcm diameter on 127 xcexcm spacing are deposited in that active area. With pins spaced apart from one another by 4.5 mm, a maximum of 16 pins can be used to fabricate such an array. The addition of even a single extra (seventeenth) pin would increase the active area beyond the desired 18 mm square. If each printing takes about a minute (where the time for each printing includes dipping the pins in the target material, contacting the pins to the substrate, and washing and drying the pins), then fabricating such an array with 16 pins will require about 21 hours (1250 separate printings of the sixteen pins are required to product 20,000 spots). Using 32 pins to fabricate the array would reduce the fabrication time in half, but would also double the footprint of the printhead and thereby double the active area of the array.
On one hand, the desire to increase the throughput of the spotting instrument suggests increasing the number of pins in the printhead to provide a high degree of parallelism in the spotting operation. On the other hand, a large number of pins in the printhead forces the microarray""s active area to be at least as large as the footprint of the pattern of pins, regardless of how many spots are in the microarray. Because the quality of the results of a microarray experiment are generally judged to be more important than the throughput, the desire for a compact array for improved control of the hybridization reaction normally dominates the decision of how many pins to use. As a result, spotting instruments are often used with less than fully populated printheads. For example, since the above-described type of array can only be printed with a maximum of sixteen pins, even if a printhead of a particular spotting instrument could accommodate 48 pins (i.e., the printhead defines 48 apertures), only 16 pins could be used and the remaining capacity to hold an additional 32 pins would be unused. So, the potential gains in throughput and productivity enabled by a high pin count often remain unrealized due to the problem of the resulting arrays being undesirably large.
It would therefore be advantageous to provide methods and systems for using large numbers of pins to produce compact microarrays.
These and other objects are provided by a pin-lifter that can selectively lift one or more pins partially out of a printhead and thereby prevent the lifted pins from contacting the substrate during any printing operation. The ability to prevent selected pins from contacting the substrate during a printing (i.e., the ability to selectively disable the printing function of selected pins) can be used, for example, to advantageously increase the rate of production of compact microarrays.
Still other objects and advantages of the present invention will become readily apparent to those skilled in the art from the following detailed description wherein several embodiments are shown and described, simply by way of illustration of the best mode of the invention. As will be realized, the invention is capable of other and different embodiments, and its several details are capable of modifications in various respects, all without departing from the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not in a restrictive or limiting sense, with the scope of the application being indicated in the claims.